Characterization of the human CIDEA promoter in fat cells

Abstract

Background: Cell death-inducing DFFA (DNA fragmentation factor-a)-like effector A (CIDEA) is a protein that regulates lipolysis in human adipocytes through cross-talk involving tumor necrosis factor-a (TNF-a). TNF-a downregulates CIDEA mRNA although it is unclear whether this is mediated through transcriptional or post-transcriptional mechanisms. CIDEA has important metabolic effects in human fat cells and genetic variations in the human CIDEA gene have been correlated to the development of obesity. However, little is known about the factors regulating CIDEA expression in human adipocytes. We set out to describe the transcriptional control of human CIDEA. Methods: A 1.1-kb genomic fragment upstream of the transcriptional start site (TSS) of human CIDEA was cloned and deletion fragments were generated. Transcriptional activity of the promoter was analyzed by luciferase reporter assays in in vitrodifferentiated human adipocytes. The effect of TNF-a was assessed in human adipocytes and murine 3T3-L1 cells transfected with deletion fragments of the CIDEA promoter. Protein-DNA interactions were analyzed by electrophoretic mobility shift assays (EMSA). Results: Basal transcriptional activity was found in a 97-bp region upstream of the TSS. We studied the effect of three common haplotypes in the promoter region but found no significant difference in transcriptional activity among them. Incubation of in vitro-differentiated human adipocytes as well as 3T3-L1 cells with TNF-a reduced the transcriptional activity of the human CIDEA promoter, demonstrating a direct effect on CIDEA transcription. EMSAs and mutational analysis indicated that this was mediated by a nuclear factor-kB (NF-kB) site at position À163/À151. Conclusion: We demonstrate that basal transcription of the human CIDEA gene is confined to the 97 first bases upstream of TSS and that TNF-a negatively regulates transcription of this gene, which at least in part involves NF-kB activation.

A Possible Inflammatory Role of Twist1 in Human White Adipocytes

“…Expression of twist1 was confirmed by in vitro translation using a TnT T7/SP6 Coupled Reticulocyte Lysate System (catalog number L5020; Promega). Gene reporter assays were performed in undifferentiated 3T3-L1 cells transfected with Lipofectamine and Plus reagent (both from Invitrogen) as described (23) using 0.2 μg of pGL2-basic or pIL6 together with either 0.2 μg of pcDNA3.1 carrying the human twist1 sequence (pTwist1) or the empty expression vector pcDNA3.1. A cytomegalovirus-β-galactosidase–containing vector (0.1 μg) was included in all transfections to control for transfection efficiency.…”

CIDE proteins and metabolic disorders

“…Cidec, a human homolog of rodent Fsp27, is induced by PPARg2 [14]. TNF-a negatively regulates transcription of Cidea through NF-kB activation [19]. In liver, Fsp27 is a direct target of PPARg [9].…”

Hypermethylation-Mediated Silencing of CIDEA, MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies

“…This means that the significance of CpG sites in the MAL and PCDH17 promoter regions was merely inferred from the negative association between the methylation state of certain CpGIs and gene expression. CiDEA promoter, on the other hand, has been partially characterized in human fat cells [ 68 ] and murine liver cells [ 69 ]. Both human and murine CiDEA promoters contain common evolutionarily conserved regions that overlap with CpGIs, and candidate TFBSs for SP1 and NF-kB have been proposed [ 68 ].…”